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. 2017 Mar 10;4(2):ENEURO.0340-16.2017.
doi: 10.1523/ENEURO.0340-16.2017. eCollection 2017 Mar-Apr.

Astrocyte Ca2+ Influx Negatively Regulates Neuronal Activity

Yao V Zhang  1 , Kiel G Ormerod  1 , J Troy Littleton  1
Affiliations

Astrocyte Ca2+ Influx Negatively Regulates Neuronal Activity

Yao V Zhang et al. eNeuro. .

Abstract

Maintenance of neural circuit activity requires appropriate regulation of excitatory and inhibitory synaptic transmission. Recently, glia have emerged as key partners in the modulation of neuronal excitability; however, the mechanisms by which glia regulate neuronal signaling are still being elucidated. Here, we describe an analysis of how Ca2+ signals within Drosophila astrocyte-like glia regulate excitability in the nervous system. We find that Drosophila astrocytes exhibit robust Ca2+ oscillatory activity manifested by fast, recurrent microdomain Ca2+ fluctuations within processes that infiltrate the synaptic neuropil. Unlike the enhanced neuronal activity and behavioral seizures that were previously observed during manipulations that trigger Ca2+ influx into Drosophila cortex glia, we find that acute induction of astrocyte Ca2+ influx leads to a rapid onset of behavioral paralysis and a suppression of neuronal activity. We observe that Ca2+ influx triggers rapid endocytosis of the GABA transporter (GAT) from astrocyte plasma membranes, suggesting that increased synaptic GABA levels contribute to the neuronal silencing and paralysis. We identify Rab11 as a novel regulator of GAT trafficking that is required for this form of activity regulation. Suppression of Rab11 function strongly offsets the reduction of neuronal activity caused by acute astrocyte Ca2+ influx, likely by inhibiting GAT endocytosis. Our data provide new insights into astrocyte Ca2+ signaling and indicate that distinct glial subtypes in the Drosophila brain can mediate opposing effects on neuronal excitability.

Keywords: Ca2+; Drosophila; GABA; GAT; Rab11; astrocyte.

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Figures

Figure 1.
Figure 1.
Near-membrane Ca2+ activity in Drosophila astrocytes. A, Single confocal plane of larvae VNC showing the astrocyte-specific expression of myrGCaMP6s under the control of Alrm-Gal4. B, B’, Time-lapse image series of two microdomain astrocyte Ca2+ transients. C, Superimposition of astrocyte Ca2+ traces (in gray) and their average (black line; n = 46 individual traces). D, Sample traces of recurring Ca2+ transients in five different areas indicated in A. Scale bars 20 µm (A) and 5 µm (B and B’).
Figure 2.
Figure 2.
Acute astrocyte Ca2+ influx through TrpA1 channels suppresses neuronal activity. A, All Alrm > TrpA1 flies became paralyzed within 30 s of exposure to 33°C, while control flies (Alrm-Gal4) show no behavioral defects. n > 80 flies per genotype; pa < 0.0001. B, Time course of paralysis in adult flies of the indicated genotypes on exposure to 33°C; repo-Gal80 suppresses the paralysis in Alrm > TrpA1 background, while elav-Gal80 has no effect. n > 70 flies for each genotype. C, Sample traces of CPG recording from larval NMJs. Postsynaptic potentials in Alrm > TrpA1 animals diminish during a temperature ramp from room temperature (22°C) to 34°C, while Alrm-Gal4 larvae maintain normal activity.
Figure 3.
Figure 3.
Suppression of Rab11 function ameliorates astrocyte Ca2+ influx-induced behavioral defects and impairs GAT trafficking. A, Quantification of paralyzed flies when exposed to 30°C, showing rab11-knockdown suppressed the paralysis in Alrm > TrpA1 flies. n > 100 flies per genotype; pb < 0.00001. B, Quantification of paralyzed flies when Alrm > TrpA1 and Alrm > TrpA1, Rab11-DN flies were transferred to 30°C. n > 45 flies per genotype; pc = 0.0095. C, Single optical section showing astrocyte cell body and neuropil in the VNCs of Alrm > TrpA1-myc (control) and Alrm > TrpA1-myc, Rab11-RNAi (Rab11-RNAi) larvae stained with antibodies against Myc (green) and Brp (magenta). Note the apposition between synapses and astrocyte processes in the neuropil. D, Quantification of relative TrpA1-Myc level, normalized to control. n = 8 individual optical planes for each groups; p d = 0.5761. E, Optical sections showing GAT localization in astrocytes of larval VNCs stained with antibodies against GAT (green) and Repo (magenta) in control and when Rab11-RNAi or Rab11-DN is expressed in astrocytes. F, Western blotting of adult heads showing the effect of Rab11-RNAi expression in astrocytes on GAT level. G, Quantification of F. n = 6 experiments; p e = 0.3125. Scale bars in C and E, 5 µm.
Figure 4.
Figure 4.
Astrocyte Ca2+ influx leads to reduced membrane GAT. A, Confocal images of larval VNC showing astrocyte membrane GAT level in control and Alrm > TrpA1 animals at room temperature (22°C) or 34°C. B, Quantification of relative GAT level shown in A, normalized to Alrm-Gal4 animals at each temperature. n = 8-10 individual optical planes for all groups; p f = 0.4974 and p g < 0.0001, respectively. C, C’, Confocal images with longer exposure showing GAT-containing vesicles (arrowheads) forming in the cytoplasm of Alrm > TrpA1 larvae exposed to 34°C. Scale bars in A and C, 5 µm.
Figure 5.
Figure 5.
Excessive GABA leads to suppression of neuronal activity. A, Quantification of paralyzed flies on exposure to 30°C. Ectopic activation of GABAergic neurons via GAD1-Gal4-induced expression of TrpA1 leads to acute suppression of neuronal activity and paralysis. B, Distinct impact of cortex glia and astrocyte Ca2+ signals on neuronal activity. Ca2+ influx in cortex glia leads to increased neural activity and seizure-like behavior, whereas enhanced astrocyte Ca2+ signal causes suppression of neuronal activity and paralysis. Together, they constitute a Ca2+-dependent glial mechanism to fine-tune neuronal function. C-C”, Model of how astrocyte Ca2+ signaling regulates neuronal activity. Astrocyte Ca2+ influx leads to acute endocytosis of membrane GAT, reduced GABA uptake, and suppression of neuronal activity. Inhibition of Rab11 function reduces the removal of membrane GAT and sustains GABA uptake, hence ameliorating the paralysis caused by astrocyte Ca2+ influx.
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